Cation exchange chromatography (methods)

ABSTRACT

The present invention provides improved methods of protein purification using CEX chromatography. Such methods generally comprise the steps of: contacting a protein of interest (e.g., an antibody) with a cation exchange resin at a first pH, that is less than the pI of the most acidic isoform of the protein of interest, such that the protein of interest binds to the resin; washing the cation exchange resin at a second pH that is greater than the first pH, but less than the pI of the most acidic isoform of the protein of interest; and eluting the protein of interest from the resin at a third pH that is about equal to or less than the first pH. The methods of the invention are particularly useful for the commercial purification of recombinant therapeutic proteins (e.g., antibodies).

BACKGROUND OF THE INVENTION

Large-scale, economic purification of proteins is an increasinglyimportant challenge for the biopharmaceutical industry. Therapeuticproteins are typically produced using prokaryotic or eukaryotic celllines that are engineered to express the protein of interest from arecombinant plasmid containing the gene encoding the protein. Separationof the desired protein from the mixture of components fed to the cellsand cellular by-products to an adequate purity, e.g., sufficient for useas a human therapeutic, is a fundamental requirement for biologicsmanufacturers. However, in therapeutic antibody purification, thecurrent industry-standard, chromatography capture resin, Protein A, isexpensive, has a relatively low throughput, and limited life cycles.

Accordingly there is a need in the art for alternative proteinpurification methods that can be used to expedite the large-scaleprocessing of protein-based therapeutics, such as antibodies, especiallydue to escalating high titers from cell culture.

SUMMARY OF THE INVENTION

The present invention provides improved methods for protein purificationusing cation exchange (CEX) chromatography. These methods generallyinvolve contacting a protein of interest (e.g., an antibody) with acation exchange resin at a first pH that is less than the isoelectricpoint (pI) of the most acidic isoform of the protein of interest, suchthat the protein of interest binds to the resin. The resin is thenwashed at a second pH that is greater than the first pH, but less thanthe pI of the most acidic isoform of the protein of interest. Theprotein is subsequently eluted from the resin at a third pH that isabout equal to or less than the first pH. This combination of higher pHwash and lower pH elution results in improved separation of the proteinof interest (e.g., an antibody) from contaminants (e.g., HCP), comparedto conventional CEX purification methods. The methods of the inventionare useful for the commercial purification of recombinant therapeuticproteins, particularly antibodies.

Accordingly, in one aspect, the invention provides a method of purifyinga protein of interest from a mixture comprising the protein of interestand one or more contaminants, the method comprising the steps of: (A)determining the pI of the most acidic isoform of the protein ofinterest; (B) contacting the protein of interest with a cation exchangeresin at a first pH that is less than the pI of the most acidic isoformof the protein of interest, such that the protein of interest binds tothe resin; (C) washing the cation exchange resin at a second pH that isgreater than the first pH, but less than the pI of the most acidicisoform of the protein of interest; and (D) eluting the protein ofinterest from the resin at a third pH that is about equal to or lessthan the first pH, thereby purifying the protein of interest.

In a particular embodiment, the first pH is between about pH 4 and aboutpH 8.2, for example, between about pH 4.5 and about pH 6.2 (e.g., at4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6.0, 6.1 or 6.2), where the pI of the most acidic isoform of theprotein of interest is between about pI 7 and about pI 9, for examplebetween about pI 7.3 and about pI 8. Exemplary pIs and pHs are shown inTable 1 and Table 2.

In certain embodiments, the second pH is greater than the first pH, butis about 0.1 to about 1.2 pH units less than the pI of the most acidicisoform of the protein of interest. In a particular embodiment, thesecond pH is between about pH 6 and about pH 8.2 (e.g., at 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8.0, 8.1 or 8.2). Exemplary pHs are shown in Table 1and Table 2.

In certain embodiments, the third pH is about equal to or less than thefirst pH. In a particular embodiment, the third pH is between about 4and about 8.2, for example, between about 4.5 and about 6.2 (e.g., at4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6.0, 6.1 or 6.2). An exemplary pH (6.2) is shown in Table 1 andTable 2.

The starting mixture may comprise any combination of proteins. In oneembodiment, the mixture comprises clarified bulk, such as a cell culturesupernatant (e.g., a mammalian, bacterial, plant or fungal cellculture). Typical cell culture supernatants include, without limitation,CHO and NSO cell cultures.

The methods of the invention can be used to purify any type of proteinfrom a mixture. In a particular embodiment, the protein is an antibody,such as a monoclonal antibody (e.g., a human, humanized or chimericmonoclonal antibody) or a fragment thereof.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows an isoelectric focus (IEF) gel of IgG1 and IgG4 humanantibodies (Humabs).

FIG. 2 shows a graph of the relationship between CEX wash pH and thelowest pI of the Humab for the Humabs listed in Table 1.

FIG. 3 shows the effects of loading, wash and elution pH on the HostCell Protein (HCP) content of the eluted Humab when the loading pH is(A) 6.2 and (B) 4.5.

FIG. 4 shows a schematic of the relationship between loading, wash, andelution pH and the contaminants present in a protein sample purified byCEX chromatography.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the terms “purifying” and “separating” are usedinterchangeably, and refer to the removal of contaminants from a mixturecontaining a protein of interest (e.g., an antibody).

As used herein, the term “protein of interest” is used in its broadestsense to include any protein (either natural or recombinant), present ina mixture, for which purification is desired. Such proteins of interestinclude, without limitation, hormones, growth factors, cytokines,immunoglobulins (e.g., antibodies), immunoglobulin-likedomain-containing molecules (e.g., ankyrin or fibronectindomain-containing molecules), and Fc-fusion proteins. The term“Fc-fusion protein”, as used herein, is meant to encompass therapeuticproteins comprising an immunoglobulin-derived moiety (i.e., an Fcmoiety) and a moiety derived from a second, non-immunoglobulin protein.

As used herein, a “mixture” comprises a protein of interest (for whichpurification is desired) and one or more contaminant, i.e., impurities.In one embodiment, the mixture is produced from a host cell or organismthat expresses the protein of interest (either naturally orrecombinantly). Such mixtures include, for example, cell cultures, celllysates, and clarified bulk (e.g., clarified cell culture supernatant).

As used herein, the term “contaminant” is used in its broadest sense tocover any undesired component or compound within a mixture. In cellcultures, cell lysates or clarified bulk (e.g., cell culturesupernatant), contaminants include, for example, host cell nucleic acids(e.g., DNA) and host cell proteins present in a cell culture medium.Host cell contaminant proteins include, without limitation, thosenaturally or recombinantly-produced by the host cell, as well asproteins related to or derived from the protein of interest (e.g.,proteolytic fragments) and other process related contaminants (e.g.,truncated and aggregated versions of the protein of interest).

As used herein, “isoform” refers to any form or variant of a protein ofinterest distinguishable by its pI. Typical isoforms include, withoutlimitation, glycoforms (i.e., proteins with differing carbohydratemoieties) and aminated variants. The “most acidic” isoform of a proteinis the isoform having the lowest pI value. Any art recognized method canbe used to determine or predict the isoelectric point (pI) of thedifferent isoforms of a protein of interest. Suitable techniquesinclude, without limitation, IEF and calculation using the known pKa ofeach charged group of the protein of interest. Previously determined orcommonly accepted pI values for the isoforms of a protein of interestcan also be used to employ the methods of the invention.

As used herein, “washing” refers to passing an appropriate bufferthrough or over a cation exchange resin.

As used herein, “eluting” refers to removing a protein of interest(e.g., an antibody) from a cation exchange resin, by altering the ionicstrength of the buffer surrounding the cation exchange resin such thatthe buffer competes with the molecule for the charged sites on the ionexchange material.

As used herein, a “cell culture” refers to cells in a liquid medium thatproduce a protein of interest. The cells can be from any organismincluding, for example, bacteria, fungus, mammals or plants. Suitableliquid media include, for example, nutrient media and non-nutrientmedia.

As used herein, the term “clarified bulk” refers to a mixture from whichparticulate matter (e.g., cells) has been substantially removed.Clarified bulk includes cell culture supernatant, or cell lysate fromwhich cells or cell debris has been substantially removed by, forexample, filtration or centrifugation.

The term “antibody” is used in the broadest sense to cover any type ofknown antibody, including, but is not limited to, monoclonal antibodies(including full length monoclonal antibodies), polyclonal antibodies,monospecific antibodies, multispecific antibodies (e.g., bispecificantibodies), immunoadhesins, antibody-immunoadhesin chimeras, humanized,human, chimeric, single-chain, synthetic, recombinant, hybrid, mutated,grafted, or in vitro generated antibodies. The antibody can be a fulllength antibody or an antibody fragment. The antibody may be selectedfrom any of the known antibody isotypes, for example, IgA, IgG, IgD,IgE, IgM. The antibody may be a monomer, dimer, or multimer (e.g., atrimer or pentamer).

An “antibody fragment” includes at least a portion of a full lengthantibody and typically an antigen binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; single-chain antibody molecules; diabodies; linearantibodies; and multispecific antibodies formed from engineered antibodyfragments.

The term “monoclonal antibody” is used in the conventional sense torefer to an antibody obtained from a population of substantiallyhomogeneous antibodies such that the individual antibodies comprisingthe population are identical except for possible naturally occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single antigenic site.This is in contrast with polyclonal antibody preparations whichtypically include varied antibodies directed against differentdeterminants (epitopes) of an antigen, whereas monoclonal antibodies aredirected against a single determinant on the antigen. The term“monoclonal”, in describing antibodies, indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, monoclonal antibodiesused in the present invention can be produced using conventionalhybridoma technology first described by Kohler et al., Nature 256:495(1975), or they can be made using recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). Monoclonal antibodies can also be isolatedfrom phage antibody libraries, e.g., using the techniques described inClackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol.222:581-597 (1991); and U.S. Pat. Nos. 5,223,409; 5,403,484; 5,571,698;5,427,908 5,580,717; 5,969,108; 6,172,197; 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915; and 6,593,081.

Monoclonal antibodies described herein include “chimeric” and“humanized” antibodies in which a portion of the heavy and/or lightchain is identical with or homologous to corresponding sequences inantibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which the hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

Chimeric or humanized antibodies can be prepared based on the sequenceof a murine monoclonal antibody prepared as described above. DNAencoding the heavy and light chain immunoglobulins can be obtained fromthe murine hybridoma of interest and engineered to contain non-murine(e.g., human) immunoglobulin sequences using standard molecular biologytechniques. For example, to create a chimeric antibody, the murinevariable regions can be linked to human constant regions using methodsknown in the art (see e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.).To create a humanized antibody, the murine CDR regions can be insertedinto a human framework using methods known in the art (see e.g., U.S.Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089;5,693,762 and 6,180,370 to Queen et al.).

The monoclonal antibodies described herein also include “human”antibodies, which can be isolated from various sources, including, e.g.,from the blood of a human patient or recombinantly prepared usingtransgenic animals. Examples of such transgenic animals includeKM-Mouse® (Medarex, Inc., Princeton, N.J.) which has a human heavy chaintransgene and a human light chain transchromosome (see WO 02/43478),Xenomouse® (Abgenix, Inc., Fremont Calif.; described in, e.g., U.S. Pat.Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 toKucherlapati et al.), and HuMAb-Mouse® (Medarex, Inc.; described in,e.g., Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295;Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon etal. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993)Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830;Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al.(1994) International Immunology 6: 579-591; and Fishwild, D. et al.(1996) Nature Biotechnology 14: 845-851, U.S. Pat. Nos. 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016;5,814,318; 5,874,299; and 5,770,429; 5,545,807; and PCT Publication Nos.WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, WO 01/14424 to Korman et al.). Human monoclonal antibodies ofthe invention can also be prepared using SCID mice into which humanimmune cells have been reconstituted such that a human antibody responsecan be generated upon immunization. Such mice are described in, forexample, U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.

As used herein the term “chromatography” refers to the process by whicha solute of interest, e.g., a protein of interest, in a mixture isseparated from other solutes in the mixture by percolation of themixture through an adsorbent, which adsorbs or retains a solute more orless strongly due to properties of the solute, such as pI,hydrophobicity, size and structure, under particular bufferingconditions of the process.

The terms “ion-exchange” and “ion-exchange chromatography” refer to achromatographic process in which an ionizable solute of interest (e.g.,a protein of interest in a mixture) interacts with an oppositely chargedligand linked (e.g., by covalent attachment) to a solid phase ionexchange material under appropriate conditions of pH and conductivity,such that the solute of interest interacts non-specifically with thecharged compound more or less than the solute impurities or contaminantsin the mixture. The contaminating solutes in the mixture can be washedfrom a column of the ion exchange material or are bound to or excludedfrom the resin, faster or slower than the solute of interest.“Ion-exchange chromatography” specifically includes cation exchange(CEX), anion exchange, and mixed mode chromatographies.

A “cation exchange resin” or “CEX resin” refers to a solid phase whichis negatively charged, and which has free cations for exchange withcations in an aqueous solution passed over or through the solid phase.Any negatively charged ligand attached to the solid phase suitable toform the cation exchange resin can be used, e.g., a carboxylate,sulfonate and others as described below. Commercially available cationexchange resins include, but are not limited to, for example, thosehaving a sulfonate based group (e.g., MonoS, MiniS, Source 15S and 30S,SP Sepharose Fast Flow™, SP Sepharose High Performance from GEHealthcare, Toyopearl SP-650S and SP-650M from Tosoh, Macro-Prep High Sfrom BioRad, Ceramic HyperD S, Trisacryl M and LS SP and Spherodex LS SPfrom Pall Technologies); a sulfoethyl based group (e.g., Fractogel SE,from EMD, Poros S-10 and S-20 from Applied Biosystems); a sulphopropylbased group (e.g., TSK Gel SP 5PW and SP-5PW-HR from Tosoh, Poros HS-20and HS 50 from Applied Biosystems); a sulfoisobutyl based group (e.g.,(Fractogel EMD SO₃ ⁻ from EMD); a sulfoxyethyl based group (e.g., SE52,SE53 and Express-Ion S from Whatman), a carboxymethyl based group (e.g.,CM Sepharose Fast Flow from GE Healthcare, Hydrocell CM from BiochromLabs Inc., Macro-Prep CM from BioRad, Ceramic HyperD CM, Trisacryl M CM,Trisacryl LS CM, from Pall Technologies, Matrx Cellufine C500 and C200from Millipore, CM52, CM32, CM23 and Express-Ion C from Whatman,Toyopearl CM-650S, CM-650M and CM-650C from Tosoh); sulfonic andcarboxylic acid based groups (e.g., BAKERBOND Carboxy-Sulfon from J. T.Baker); a carboxylic acid based group (e.g., WP CBX from J. T. Baker,DOWEX MAC-3 from Dow Liquid Separations, Amberlite Weak CationExchangers, DOWEX Weak Cation Exchanger, and Diaion Weak CationExchangers from Sigma-Aldrich and Fractogel EMD COO—from EMD); asulfonic acid based group (e. g., Hydrocell SP from Biochrom Labs Inc.,DOWEX Fine Mesh Strong Acid Cation Resin from Dow Liquid Separations,UNOsphere S, WP Sulfonic from J. T. Baker, Sartobind S membrane fromSartorius, Amberlite Strong Cation Exchangers, DOWEX Strong Cation andDiaion Strong Cation Exchanger from Sigma-Aldrich); and a orthophosphatebased group (e.g., P11 from Whatman).

Mixtures Containing a Protein of Interest

The methods of the invention can be applied to purify one or moreprotein(s) of interest from any mixture containing the protein(s). Inone embodiment, the mixture is obtained from or produced by living cellsthat express the protein to be purified (e.g., naturally or by geneticengineering). Methods of genetically engineering cells to produceproteins are well known in the art. See e.g., Ausabel et al., eds.(1990), Current Protocols in Molecular Biology (Wiley, New York) andU.S. Pat. Nos. 5,534,615 and 4,816,567, each of which is specificallyincorporated herein by reference. Such methods include introducingnucleic acids that encode and allow expression of the protein intoliving host cells. These host cells can be bacterial cells, fungalcells, or, preferably, animal cells grown in culture. Bacterial hostcells include, but are not limited to E. coli cells. Examples ofsuitable E. coli strains include: HB101, DH5a, GM2929, JM109, KW251,NM538, NM539, and any E. coli strain that fails to cleave foreign DNA.Fungal host cells that can be used include, but are not limited to,Saccharomyces cerevisiae, Pichia pastoris and Aspergillus cells. A fewexamples of animal cell lines that can be used are CHO, VERO, DXB11,BHK, HeLa, Cos, MDCK, 293, 3T3, NS0 and WI138. New animal cell lines canbe established using methods well know by those skilled in the art(e.g., by transformation, viral infection, and/or selection). In otherembodiments, the protein of interest (e.g., an antibody) is produced ina CHO cell (see, e.g., WO 94/11026). Various types of CHO cells areknown in the art, e.g., CHO-K1, CHO-DG44, CHO-DXB11, CHO/dhfr⁻ andCHO-S.

Preparation of mixtures initially depends on the manner of expression ofthe protein. Some cell systems directly secrete the protein (e.g., anantibody) from the cell into the surrounding growth media, while othersystems retain the antibody intracellularly. For proteins producedintracellularly, the cell can be disrupted using any of a variety ofmethods, such as mechanical shear, osmotic shock, and enzymatictreatment. The disruption releases the entire contents of the cell intothe homogenate, and in addition produces subcellular fragments which canbe removed by centrifugation or by filtration. A similar problem arises,although to a lesser extent, with directly secreted proteins due to thenatural death of cells and release of intracellular host cell proteinsduring the course of the protein production run.

In one embodiment, cells or cellular debris are removed from themixture, for example, to prepare clarified bulk. The methods of theinvention can employ any suitable methodology to remove cells orcellular debris, including, centrifugation, tangential flow filtrationor depth filtration.

Protein Purification

The methods of the invention provide improved techniques for CEXpurification of a protein of interest (e.g., an antibody) from amixture. These methods generally comprise the steps of: (A) contactingthe protein of interest with a cation exchange resin at a first pH thatis less than the pI of the most acidic isoform of the protein ofinterest, such that the protein of interest binds to the resin; (B)washing the cation exchange resin at a second pH that is greater thanthe first pH, but less than the pI of the most acidic isoform of theprotein of interest; and (C) eluting the protein of interest from theresin at a third pH that is about equal to or less than the first pH.However, the skilled artisan will appreciate that additionalpurification can be performed before, after or in between the steps ofthe aforementioned method. The combination of higher pH wash and lowerpH elution employed in the methods of the invention results in efficientseparation of the protein of interest (e.g., an antibody) fromcontaminants (e.g., HCP), compared to that achieved using conventionalCEX purification methods.

TABLE 1 pI Ranges and CEX Load, Wash and Elution pHs For Eight HumabsΔpH lowest CEX wash (lowest pI-wash CEX load CEX Product pI range pI pHpH) pH elution pH Humab-1 8.60-8.81 8.6 8.2 0.4 6.2 6.2 Humab-28.19-8.50 8.19 7.5 0.69 6.2 6.2 Humab-3 8.68-8.83 8.68 7.5 1.18 6.2 6.2Humab-4 7.96-8.39 7.96 7.5 0.46 6.2 6.2 Humab-5 7.57-8.18 7.57 7.2 0.376.2 6.2 Humab-6 8.14-8.49 8.14 7.8 0.34 5.7 6.2 Humab-7 8.28-8.78 8.287.2 1.08 6.2 6.2 Humab-8 * 7.33-8.21 7.33 7.1 0.23 6.2 6.2 * Humab-8 isa IgG4 molecule

Table 1 shows exemplary binding, wash and elution pHs for the CEXpurification of eight human monoclonal antibodies according to themethods of the invention. FIG. 4 schematically shows particularembodiments of the methods.

Any methods can be used to determine the pI of the most acidic isoformof the protein of interest (e.g., an antibody). Such methods include,without limitation, isoelectric focusing (IEF) techniques, and manual orin silico calculation using the known pKas of the charged groups of theprotein of interest. An example of IEF determination of the pI of thedifferent isoforms of eight human antibodies is shown in FIG. 1.However, the skilled artisan will appreciate that, if the pI values forthe various isoforms of a protein are already known, it is not necessaryfor them to be determined again in order to practice the methods of theinvention.

Binding of the protein of interest (e.g., an antibody) to a cationexchange resin can be performed at any pH below the pI of the mostacidic isoform of the protein to be purified. In particular embodiments,the protein of interest (e.g., an antibody) is bound to the resinbetween about pH 4 and about pH 8 (e.g., about pH 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, and 8). In one exemplified embodiment, the protein ofinterest (e.g., an antibody) is bound to the resin at about pH 6.2. Inanother exemplified embodiment, the protein of interest (e.g., anantibody) is bound to the resin at about pH 4.5.

Once the protein (e.g., an antibody) is bound to the cation exchangeresin, contaminants (e.g., HCP) are removed by washing the resin with abuffer at a second pH that is greater than the first pH, but less thanthe pI of the most acidic isoform of the protein. The optimal pH forwashing the resin can be determined empirically for each protein ofinterest by monitoring the purity and yield of the purified protein. Incertain embodiments, the resin is washed at a pH of between about 0.1and about 2.0 pH units below the pI of the protein of interest, morepreferably between about 0.1 and about 1.2 pH units below the pI (e.g.,about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 and 1.1 pH units belowthe pI). Table 1 shows exemplary wash pHs relative to pI for the CEXpurification of nine human monoclonal antibodies according to themethods of the invention. The wash buffer can be augmented withdetergents or surfactants (e.g., polysorbate) to further removecontaminants, e.g., DNA and endotoxin contaminants.

After washing the cation exchange resin, the protein of interest iseluted using a buffer with a pH about equal to, or less than, thebinding pH. In certain embodiments, the elution pH (third pH) is betweenabout 0 and about 3 pH units below the binding pH (first pH). In oneparticular embodiment, the elution pH is about pH 6.2. In anotherparticular embodiment, the elution pH is about pH 4.5. In general, theelution is facilitated by increasing the ionic strength of the elutionbuffer relative to the binding buffer, for example, by the addition of asalt (e.g., sodium chloride) to the elution buffer. In addition, apolyether (e.g., polyethylene glycol) can be added to the elution bufferto reduce protein aggregation and the formation of higher molecularweight species.

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference in their entireties.

EXAMPLE 1

In this Example a Humab (Humab-1 in table 1) was purified by CEXchromatography using different combinations of load, wash and elutionpHs. In all experiments the CEX resin (Poros 50HS (Applied Biosystem))was packed in a 1 mL column (Φ0.5 cm×H 5 cm).

In one set of experiments (FIG. 3A), a CEX column was equilibrated withSodium Phosphate buffer at pH 6.2 and 5.8 mS/cm. A solution of Humab-1was adjusted to pH 6.2 and 5.8 mS/cm, and loaded onto the column at aconcentration of 15 mg/mL. The column was then washed with SodiumPhosphate and Sodium Chloride dual buffer at pH 7.2 or Sodium Phosphatebuffer at pH 8.0. After the wash step, the Humab was eluted from thecolumn at pH 8.2, 8.0, 7.5, 7.2, 6.2, or 4.5 with Sodium Phosphatebuffer containing sufficient Sodium Chloride to reach the appropriateconductivity for elution.

In another set of experiments (FIG. 3B), a CEX column was equilibratedwith Sodium Citrate and Sodium Phosphate dual buffer at pH 4.5 and 2mS/cm. A solution of Humab-1 was adjusted to pH 4.5 and 2mS/cm, andloaded onto the column at a concentration of 15 mg/ml. The column wasthen washed with Sodium Phosphate and Sodium Chloride dual buffer at pH7.2 or Sodium Phosphate buffer at pH 8.0. After the wash step, the Humabwas eluted from the column at pH 6.2, or 4.5 with Sodium Phosphatebuffer containing sufficient Sodium Chloride to reach the appropriateconductivity for elution.

These data demonstrate that if the elution pH is lower than wash pH,preferably about equal to or below the load pH, the greatest removal ofhost cell contaminants will result. It also illustrates that the bestpurity of the Humab can be achieved when the wash buffer pH is keptbelow the pI of the most acidic isoform of the molecule, but as high aspossible without washing the Humab off the column.

EXAMPLE 2

In this Example IgG1 (Humab-1 in table 1) and IgG4 (Humab-8 in table 1)Humabs were purified using the methods of the invention (see Table 2). ACEX column was equilibrated to pH 6.2. Unpurified bulk containing eitherHumab-1 or Humab-8 was buffer-exchanged to the same pH and ionicstrength as the column equilibration buffer and loaded onto a cationexchange column. The column was then washed with Sodium Phosphate bufferat a pH just below the pI of each Humab. After the wash step, the Humabswere eluted from the column with a buffer comprising either 35 mM SodiumPhosphate, 75 mM NaCl at pH 6.2 (Humab-1) or 75 mM Sodium Phosphate, pH6.2 (for Humab-8).

The data in Table 2 show that the CEX chromatography methods of theinvention resulted in about a 1000-fold reduction in the HCP contaminantfor both Humab-1 and Humab-8.

TABLE 2 IgG1 and IgG4 Humab Purification Using the Methods of theInvention Humab-1 (IgG1) Humab-8 (IgG4) (lowest pI 7.96) (lowest pI7.33) CHO HCP DNA CHO HCP DNA Step Solution (ng/mg) (pg/mg) StepSolution (ng/mg) (pg/mg) Equilibration 70 mM Sodium Equilibration 35 mMSodium Phosphate, pH 6.2 Phosphate, pH 6.2 Load Unprocessed bulk 2.6 ×10⁴ 2.6 × 10⁴ Load Unprocessed bulk 108,615 808 (pH 6.2) (pH 6.2) PostLoad 10 mM Sodium Post Load 10 mM Sodium Wash Phosphate, 0.1% WashPhosphate, 0.5% Polysorbate 80, pH 7.5 Polysorbate-80, pH 7.1 Elution 35mM Sodium 21.9 39.5 Elution 75 mM Sodium 1,294 25 Phosphate, 75 mMPhosphate, pH 6.2 NaCl, pH 6.2

EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

All patents, pending patent applications, and other publications citedherein are hereby incorporated by reference in their entireties.

REFERENCES

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1. A method of purifying a protein of interest from a mixture comprisingthe protein of interest and one or more contaminants, comprising: (a)determining the pI of the most acidic isoform of the protein ofinterest; (b) contacting the protein of interest with a cation exchangeresin at a first pH that is less than the pI of the most acidic isoformof the protein of interest, such that the protein of interest binds tothe resin; (c) washing the cation exchange resin at a second pH that isgreater than the first pH, but less than the pI of the most acidicisoform of the protein of interest; and (d) eluting the protein ofinterest from the resin at a third pH that is about equal to or lessthan the first pH, thereby purifying the protein of interest.
 2. Themethod of claim 1, wherein the first pH is about 6.2.
 3. The method ofclaim 1, wherein the second pH is about 0.1 to about 1.2 pH units lessthan the pI of the most acidic isoform of the protein of interest. 4.The method of claim 1, wherein the third pH is about 4.5.
 5. The methodof claim 4, wherein the mixture comprises clarified bulk.
 6. The methodof claim 4, wherein the clarified bulk comprises a cell culturesupernatant.
 7. The method of claim 6, wherein the supernatant is from amammalian, bacterial or fungal cell culture.
 8. The method of claim 6,wherein the supernatant is from a Chinese Hamster Ovary (CHO) cellculture.
 9. The method of claim 8, wherein the contaminant comprisesfragments or aggregates of the protein of interest.
 10. The method ofclaim 9, wherein the protein of interest is an antibody.
 11. The methodof claim 10, wherein the antibody is a monoclonal antibody.
 12. Themethod of claim 11, wherein the monoclonal antibody is selected from thegroup consisting of a human, humanized and chimeric antibody.